Intra-mPFC injection of sodium butyrate promotes BDNF expression and ameliorates extinction recall impairment in an experimental paradigm of post-traumatic stress disorder

Objective(s): Therapeutic strategies that facilitate extinction are promising in the treatment of post-traumatic stress disorder (PTSD). Brain-derived neurotrophic factor (BDNF) has a crucial role in neural plasticity, a process needed for the retention of fear extinction. In this study, we investigated the effects of local administration of a histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu), on BDNF transcription and behavioral markers of extinction in the single prolonged stress (SPS) model of PTSD. Materials and Methods: NaBu was infused into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) of male rats. The freezing response was recorded as the criterion to assess fear strength on the day of extinction as well as 24 hr later in the retention test. Other behavioral tests were also measured to evaluate the anxiety level, locomotor activity, and working memory on the retention day. HDAC activity and BDNF mRNA expression were evaluated after the behavioral experiments. Results: NaBu facilitated the recall of fear extinction in SPS rats (P<0.0001). SPS rats had higher HDAC activity (P<0.0001) and lower BDNF expression (P<0.05) than non-SPS animals. Also, anxiety was higher in the SPS group (P<0.0001), but locomotor activity (P=0.61) and working memory (P=0.36) were not different between SPS and Non-SPS groups. Conclusion: Our findings provide evidence that the mechanism of action of NaBu in the improvement of extinction recall is mediated, in part, by enhancing histone acetylation and reviving BDNF expression in IL.


Introduction
Post-traumatic stress disorder (PTSD) is a debilitating stress-related disorder that is characterized by an exaggerated fearful response to reminders of a traumatic event (1). Single prolonged stress (SPS) is a widely used rodent model of PTSD that recapitulates many of the neurobiological and behavioral alterations observed in PTSD patients (2). It is shown that SPS rats have deficits in retention of extinct fearful memories acquired in classical conditioning paradigms (3). Clinical studies have found the same maladaptation in humans (4)(5)(6)(7). In conditioning paradigms, subjects learn, through repeated exposure, that a neutral or conditioned stimulus (CS) is followed by an aversive or unconditioned stimulus (US). Thus, they show a fearful response, like freezing, when CS is used alone. The fearful response will gradually fade if CS is repeatedly presented without US, a process which is called extinction (8). Extinction is a popular measure for investigation of the neurobiology of PTSD in preclinical studies (9). It is found that some PTSD symptoms occur when there are deficits in certain neural networks responsible for extinction impairments (10). Three cerebral areas, including the Medial Prefrontal Cortex (mPFC), amygdala, and hippocampus are key regulatory regions for conditioning and extinction in the brain (11). According to a well-studied model, some featured characteristics of PTSD, like exaggerated startle responses or increased freezing behavior are due to reduced amygdala inhibition by mPFC (12)(13)(14)(15)(16). Two sub-regions of mPFC are found to be involved in the control of fear behavior. The infralimbic (IL) division has a suppressive role in the expression of fear responses through inhibition of the central nucleus of the amygdala, whereas the prelimbic (PL) division seems to enhance the expression of fear memory, presumably, by activating neurons in the basal amygdala (17)(18)(19). At molecular levels, changes in behavioral parameters stem from the effects of stress on synaptic structure and function (20). Previous studies show that for the proper establishment of extinction memory, neural plasticity is a prerequisite in mPFC (9,21) and basolateral amygdala (22). Synaptic plasticity is a process that changes the connective strength between two synapses (23). Neurotrophins are a family of proteins that, among other factors, play important roles in the plasticity of synaptic transmission. The most related neurotrophin studied so far, is the brain-derived Sodium butyrate, BDNF, and extinction recall in a PTSD model Mohammadi-Farani et al. neurotrophic factor (BDNF). The role of BDNF in the extinction of fear memories is reviewed elsewhere (24). Bredy et al. (2007) found that extinction is accompanied by increased expression of BDNF mRNA in the PFC (25). The role of BDNF in the prefrontal lobe is further confirmed by the results of a recent study that showed that infusion of proBDNF into the IL prior to extinction training enhances the learning of extinction in a rat model of conditioning (26). Histone acetylation plays an important role in the control of BDNF gene transcription. Histones are a group of chromatin proteins that have five major families: H1/ H5, H2A, H2B, H3, and H4. Alterations of the acetylated state in certain histones are crucial in controlling the transcription of some eukaryotic genes. Histone acetylation or deacetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively (27). Histone deacetylase inhibitors like trichostatin A, sodium butyrate (NaBu), and valproic acid (VPA) are widely used in conditioning and extinction experiments (25,(28)(29)(30). Systemic administration of valproic acid has induced BDNF mRNA levels within mPFC after an extinction procedure. This was associated with enhanced acetylation of histone 4 around the promotor 4 of the gene (25). Exposure of rats to the SPS protocol also modulates histone acetylation and changes BDNF transcription in the hippocampus of conditioned rats (31,32). Based on the above, we hypothesized that transcriptional changes in the BDNF gene that are due to modification of histone acetylation in mPFC might be an underlying cause for extinction deficits in PTSD subjects. Therefore, in the current study, we examined the effects of bilateral infusion of NaBu into the IL of SPS rats, on extinction learning and extinction recall in a cued fear conditioning model.

Animals
All experiments were carried out using male Wistar rats (7-10 weeks old, 230-260 g body weight). Animals were housed at a controlled temperature (22 ± 2 °C) and on a 12/12-hr light/dark cycle. Behavioral tests were performed between 08:00 am and 1:00 pm. food and tap water were provided ad libitum. Animals were handled according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition. Washington DC, National Academies Press, US).

Drugs
Sodium butyrate was purchased from sigma-Aldrich (Sigma-Aldrich, Taufkirchen, Germany; 50, 250 mM). It was dissolved and diluted in 0.9% saline solution and injected in a volume of 0.5 µl per side with an infusion rate of 0.5 µl/ min. Doses were selected based on previous research (33,34) and a pilot study in our laboratory.

Experimental groups
Six groups of rats were used in our experiments. Three groups (n = 16) underwent the SPS procedure (SPS groups) and received different doses (0, 50, and 250 mM) of NaBu before the training session ( Figure 1). After training, a subset of rats (n = 8), for each group, were sacrificed and used in the molecular experiments. The rest of the rats were tested through more behavioral procedures on the extinction day. Three groups of rats (n = 16) were non-SPS groups that were treated similarly to the SPS groups except that they were not exposed to the SPS procedure.

Single prolonged stress procedure
SPS was performed according to the procedure described above (35). It began with a 2-hr restraining period in which rats were immobilized for 2 hr in a clear polyethylene cone. Next, animals were immediately put into a pool of water (circular, 60 cm diameter, 22 °C) and forced to swim for 20 min. After a 15-min rest interval, rats were exposed to diethyl ether until they briefly lost their consciousness. Finally, they were returned to their cages and remained undisturbed for 7 days before more tests ( Figure 1).

Stereotaxic surgery
Surgery was performed as described elsewhere (36). Briefly, guide cannulas (stainless steel; 10 mm long, 22 gauges) were inserted bilaterally into the IL (AP: +3 mm, ML: ±0.8 mm, DV: -4 mm) (37) subregion of the brain while the animals were anesthetized with pentobarbital sodium solution (50 mg/kg, IP). The cannula was fixed to the skull, with dental cement, so that its tip was 1 mm above IL. After a 7-day period of recovery, the animals were used for further experiments (Figure 1). To confirm the precision of the position of the cannula, methylene blue (4%, 0.5 µl) was microinjected into the IL region at the end of the behavioral experiments. Brains were removed and kept in formalin (10%) for 3 days before they were sectioned and examined for cannula placement ( Figure 2).

Conditioning apparatus
A fear conditioning apparatus, Ugo Basile (model 46002) controlled by AnyMaze software (Version 2.1; Ugo Basile), was used to study the cued fear in rats. It consisted of the main chamber, conditioning (context A), and a test (context B) box. Context A was a Plexiglas container (26×26×30 cm) that was settled on a stainless-steel electrified grid floor. The extinction training and extinction tests were carried out in a different box (context B) which had similar outside dimensions to context A but was changed internally by putting thin plastic inserts of different shapes and colors along its interior walls. Context B was further contrasted to the original training context (context A) by using a 0.5% peppermint solution for its aromatization. The main chamber had a white fluorescent lamp on top for the presentation of the conditioned stimulus (CS) and an LED lamp for providing a basal dim light.

Conditioning and extinction protocols
Conditioning took place in the context A of the apparatus. In the conditioning phase, animals received 7 pairings of the conditioned stimulus (CS), flashing house light (500 ms on/off, 8 lx, 30 sec), which was co-terminated by a foot-shock (0.7 mA, 2 s), as the unconditioned stimulus (US). The interval between each CS-US pairing was 2 min. One day later, NaBu was administered to the rats 2 hr before the extinction training. Extinction consisted of 8 cycles of CS (duration: 30 sec, interval: 90 sec) without presenting US in context B of the apparatus. More behavioral tests were performed after the extinction test was over. Twentyfour hours later, animals were tested for fear expression in context B while they were presented with a 2-min CS alone ( Figure 1).

Elevated plus maze test
This is a plus-shaped maze made of two wooden open arms (60 × 12 ×1 cm) that are perpendicular to two closed arms (60 × 12 × 35cm). The apparatus was fixed 50 cm above the ground. The animals were put in the middle square of the crossing arms with their faces towards an open arm. Movements of the rats in the maze were recorded in a 10 min period by the EthoVision XT8 video tracking system (Noldus, Netherlands). The percentage of time spent in the open arms and the number of entries into the open arms were calculated by the software. These indices are decreased as the anxiety level increases in the rodents (38).

Open field test
The open field (OF) test is used to evaluate the locomotor activity and anxiety levels in the animals. The apparatus consists of a 75 cm × 75 cm × 40 cm enclosed square arena whose floor is divided into 25 equal squares (15 cm×15 cm). Animals are left separately in the field for a 5-min acclimatization period. After that, they were allowed for another 5 min free exploration during which the number of square crossings, as an index for locomotor activity, and the time spent in the center square, as an index for anxiety, were recorded (39).

Morris water maze test
In this test, we used a black water tank (circular, diameter 120 cm, height 80 cm) as the water maze. The tank was filled with lukewarm tap water (23 ± 3 °C) to a height of 50 cm. There was a Plexiglas scape platform (circular, diameter 20 cm) in the center of an arbitrarily defined southwest quadrant of the pool that was submerged 2 cm below the surface of the water. Each rat underwent training and test sessions in the experiment. In the training session, the rat was released into the water in the opposite quadrant of the platform quadrant such that its head was towards the pool wall. During a 120 sec period, if rats could find the platform, they were allowed to stay for 10 sec and if they were not successful in locating it, they were gently guided to the platform and let stay on it for 10 sec. Next, the animals were towel-dried and kept in a warm cage until the test session. After 2 hr, the rats were placed in the same position of the pool and allowed to find the platform in 120 sec. The movement of the rats was recorded by a camera on top of the pool and the videos were analyzed by the EthoVision XT6 tracking system (Noldus Information Technology, Wageningen, Netherlands). Attenuation of the time needed to find the platform in the test session was considered an improvement in the working memory of the animals (40).

Histone deacetylase activity measurement
We evaluated the effects of NaBu treatment on HDAC activity 4 hr after drug treatment. Briefly, after decapitation, brains were removed, and the right and left IL were isolated on an ice-chilled petri dish. An ultrasonic homogenizer (Bandelin Electronic GmbH, Germany) was used to prepare homogenized tissues in 1 ml of 0.05 M phosphate buffer (pH=7). Samples were mixed to create 4 pools of 2 samples.

Real-time PCR
Samples for the PCR were prepared 2 hr after the training test. Rats were decapitated and after removal of the brains, IL was isolated for PCR experiments. Four pools of 2 samples per pool were made from the homogenate samples described previously. Total RNA was extracted using the Tissue RNA Extraction Kit (Roche Applied Science, USA) as directed by the manufacturer's instructions. We used a PrimeScript RT reagent Kit (Takara, Shiga, Japan) to make cDNAs from the RNA. The obtained cDNA was amplified using a StepOnePlus real-time PCR System (Applied Biosystems, USA). The internal standard was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For BDNF target gene assessment, primer sequences were as follows: GAPDH fwd: 5´-tac-cag-ggc-tgc-ctt-ctc-ttg-3´, GAPDH rev: 5´-gga-tct-cgc-tcc-tgg-aag-atg-3´. BDNF fwd: 5´-tct-acg-aga-cca-agt-gta-atc-cc-3´, BDNF rev: 5´-tct-atcctt-atg-aac-cgc-cag-c-3´. Each reaction had a total volume of 25 µl that contained: primers; 1 µl/primer, Power SYBR

Statistical analysis
For the extinction training tests, the two-way repeated measure (RM) ANOVA was used to evaluate the difference between the means among different groups. Data from the extinction test, EPM, OF and HDAC activity tests were compared by the two-way ANOVA with dose and stress as the main effects of comparison. A three-way analysis of variance was used to compare the working memory among different groups with treatment, stress, and training states as the main factors of comparison. We used GraphPad Prism 8.0.2 (GraphPad Software, Inc., CA, USA) to analyze data from the above experiments. In real-time PCR, we investigated the expression levels of BDNF using REST 2008 (V 2.0.7) as our software tool. The relative quantification method (2 -ΔΔCT ) was used to calculate the fold change expression ratio of BDNF transcription (41).

Effect of SPS and NaBu on extinction learning in SPS-rats
One day after conditioning, extinction training was carried out in a different environment (context B) from the conditioning stage. The training was carried out in 8 cycles of US free trials. Results of a two-way repeated measure ANOVA showed that for both the SPS and Non-SPS animals, there was a significant difference among different cycles of training

Effect of SPS and NaBu on extinction recall in SPS-rats
Two days after fear conditioning, rats were examined for freezing behavior in context B of the apparatus. The results showed that freezing was significantly higher in the SPS

Effect of SPS and NaBu on the elevated plus maze test in SPS rats
Levels

Effect of SPS and NaBu on the open field test in SPS rats
As described before the test was performed to measure the locomotor activity and anxiety level in the animals ( Figure 6). There was a significant difference for the main effect of stress in the amount of time spent in the center of

Effect of SPS and NaBu on working memory in SPS rats
In the MWM test, a three-way ANOVA test was used to evaluate the effects of stress, dose, and initial training on the latency to reach the platform.

Effects of SPS and NaBu on HDAC activity
The effects of NaBu treatment on HDAC activity in mPFC are depicted in Figure 8.

Effect of SPS and NaBu on BDNF gene expression in SPS rats
Exposure to stress resulted in a significant reduction of BDNF transcription is SPS rats  (Figure 9). Data obtained from these studies will help us better interpret the results of the behavioral experiments and provide evidence for the probable mechanisms of stress in the neurobiology of PTSD.

Discussion
The purpose of this study was to evaluate the effect of NaBu on HDAC activity and BDNF expression in mPFC and to find its possible effects on the extinction process in rats. Our main findings are (1) Local pre-extinction treatment with NaBu facilitates extinction learning in SPSrats as opposed to the non-SPS group in a cued conditioning model, (2) NaBu reverses the impairment of extinction recall in SPS-exposed animals in a dose-dependent way, (3) the expression of BDNF mRNA is increased in SPS rats in response to NaBu administration. Impairment of inhibitory learning is a central dysfunctional element in the neurobiology of PTSD. It means that PTSD patients have difficulty learning to dissociate certain reminding stimuli with a past traumatizing event (42). This deficiency is mostly investigated in extinctionrelated studies in preclinical settings (7). Single prolonged stress (SPS) is a widely used animal model to study PTSD. In this study, we have investigated the effects of pre-extinction IL microinjection of a histone deacetylase inhibitor (NaBu) on behavioral responses and molecular modifications in the region. Previous studies show that enhancement of histone acetylation in the PFC and amygdala are essential epigenetic regulators for a proper extinction process (43,44). Further immunohistochemical research suggested that extinction learning activates neurons and increases acetyl H3/H4 expression in the IL region of mPFC (45). In accordance with these studies, our experiments show that microinjection of sodium butyrate into the IL has two main extinction-related behavioral consequences: (1) it enhances (at 250 mM) the extinction learning process in the SPS group, as evident from reduced freezing in the last two cycles of extinction training (Figure 3), and (2) it reverses the impairment of extinction recall one day after extinction training in a dose-dependent manner (Figure 4). At molecular levels, a dose-dependent pattern is seen in the inhibitory action of NaBu on HDAC activity (Figure 8) in the SPS group, which is supportive of an HDAC-dependent action of the drug. Interestingly, NaBu has no effect on extinction and does not change HDAC activity in the non-SPS (normal) group. Therefore, it seems that exposure to single prolonged stress is the underlying cause for these molecular and behavioral changes. It is widely shown that systemic administration of some HDAC inhibitors like sodium butyrate (30), valproic acid (25,28,46), vorinostat (47), RGFP963 (48), and MS-275 (46) have enhancing effects on the learning or retention of extinction memory in normal rodents. The inconsistency between our results and these observations may be due to different routes of drug administration. In systemic treatments, multiple brain areas are exposed and regarding the complex nature of extinction memory, the interplay of multiple cerebral nuclei ultimately determines the outcome. However, we observed a declining trend in the freezing time in the recall test of the non-SPS group (Figure 4) which was associated with a similar trend in HDAC activity ( Figure 8). Therefore, it is also probable that we could see significant effects if higher doses of NaBu were used in the non-SPS rats.
The determinant role of BDNF in the extinction of fearful memories is shown in several studies. These studies have particularly focused on the BDNF effect in the hippocampus (49,50), amygdala (51)(52)(53), and prefrontal cortex (54)(55)(56). A recent study shows that SPS rats have lower BDNF protein levels in their IL. This study also finds that infusion of BDNF into IL, 1 hr prior to extinction training, ameliorates the impaired extinction in the retention test (54). In concert with this study, our results indicate that BDNF gene transcripts are less expressed in the IL of the SPS group (Figure 9), and impairment of extinction is recovered at a dose that brings   (Figures 3, 4). Regarding the influence of stress on BDNF gene transcription, Takei et al. (2001) have shown that exon-specific m-RNA levels of BDNF are increased after contextual fear conditioning in the hippocampus of SPS rats (32). These findings suggest that stress-induced transcriptional changes in the BDNF gene might be an underlying factor, and hence a good treatment target in patients with PTSD. BDNF transcription is regulated by transcriptional factors and epigenetic mechanisms (57). As an epigenetic regulatory mechanism, acetylation of H4 in chromatins at BDNF promoter IV and its mRNA transcripts were increased in the prefrontal cortex when rats underwent the extinction protocol (25). We have found that contrary to the non-SPS group, BDNF transcription is significantly increased in the SPS group after NaBu treatment (Figure 9). In fact, it seems that treating animals with SPS has induced a state in which they have become more sensitive to the effects of the drug. These results collective with the results obtained from the enzymatic assessment (i.e., enhanced reduction of HDAC activity in the SPS group) imply that reduced BDNF transcription in the SPS group is secondary to enhanced deacetylation of BDNF-regulating histone sites. It appears that NaBu enhancement of extinction recall involves a reverse process in the IL.
Additional behavioral tests were performed to find if SPS and NaBu would affect other confounding animal behaviors. The results of the EPM in conjunction with the OF test disclose that there is a significant difference in the main effect of SPS-state in that the SPS group has higher anxiety scores in both tests (Figures 5, 6B). Other investigators have also reported the association between anxiety and SPS (58)(59)(60)(61)(62)(63)(64). Increased anxiety is a common complication in many patients with PTSD (65). In preclinical settings, it induces a sustained defensive state that is manifested as anxietylike behaviors in different models (66). The result of the OF test also shows that the overall activity of the rats in the SPS group is not different from the non-SPS one ( Figure  6A), which means that the differences reported in the EPM or extinction tests are not confounded by the locomotor activity of the animals.
Working memory is a cognitive process that is one of the main functions of mPFC (67). It is believed that working memory and extinction have shared neurocircuitry in the brain (68,69) and most probably, mPFC-hippocampus connectivity is involved (70). Therefore, we assumed that the effect of SPS or drug on the working memory might be similar to what was seen in the extinction test. The MWM test reveals that SPS exposure and dose of NaBu do not change the working memory effect (Figure 7). It is concluded that SPS and drug treatment have exclusive effects on fear memory traces and have no effects on working memory circuits.

Conclusion
The results of the current investigation provide evidence that SPS-induced impaired extinction retention is accompanied by higher HDAC activity and reduced BDNF transcription in IL. This impaired retention is rescued by local pre-extinction administration of NaBu that leads to the reversal of HDAC activity and BDNF transcription. It is postulated that increased acetylation of histones near BDNF gene sequences, and the resultant up-regulation of BDNF expression, are the basis of NaBu-induced enhancement of extinction retention in PTSD rats.